Human keratinocytes produce many components of the plasminogen activator (PA) proteolytic cascade, including 2 enzymes (urokinase type PA (uPA) and tissue type PA (tPA)), two inhibitors types 1 and 2), and a receptor for uPA. In the epidermis, as in many other tissues, the PA cascade appears to be a physiological mechanism for the rapid generation of highly regulated proteolytic activity. Although the functions of this proteolytic activity in epidermis remain unclear, expression of the various PA components varies with the migratory and differentiation state of the keratinocyte. Specifically, uPA and PAI-1 are associated with migrating keratinocytes; in contrast, tPA and PAI-2 are found preferentially in the-more stratified, differentiated cells. The present proposal seeks to define functional aspects of the PA cascade in keratinocyte migration and differentiation. Several experimental systems will be used: 1.) Keratinocytes from mice that express the human plasminogen activator inhibitor type l (PAI-1) gene will be isolated and propagated; expression of the PAI-1 transgene followed by deposition of PAI-1 onto the extracellular matrix can be induced with ZnCl-2. Transgenic and wild type keratinocytes will be analyzed for migration using single cell and epithelial sheet assays and for stratification and differentiation parameters using submerged culture and the skin equivalent. 2.) A human keratinocyte line will be transfected with constructs that allow the over-expression and under-expression of tPA or plasminogen activator inhibitor type 2 (PAI-2). Stratification and differentiation parameters will then be compared in transfectants and parent cell lines, using routine submerged culture, skin equivalent culture at the air-liquid interface, and grafting of cultured cells onto nude mice. 3.) The involvement of the PA cascade will be examined during the stages of the murine hair cycle, a physiological model of invasion, tissue remodeling, and cellular degeneration. Expression of the PA cascade components will be evaluated at the mRNA, protein, and activity levels during the growth, degenerative, and resting phases. An organ culture model of follicular growth will be used to test the necessity of PA activity in this process. These studies will help to define the involvement of proteolytic activity in keratinocyte migration and differentiation, two processes that are essential to normal epidermal renewal, regeneration, and barrier function. Keratinocyte migration is a crucial early component of normal wound repair. Keratinocyte differentiation continues throughout life and is disrupted in several cutaneous disorders, including psoriasis. An in depth understanding of keratinocyte migration and differentiation, including the role of proteolysis therein, is thus essential if we are to understand how the skin performs its vital functions.